Detection of Haplotypes Associated with Prenatal Death in Dairy Cattle and Identification of Deleterious Mutations in GART,SHBG and SLC37A2

The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10⁻⁴) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle.     

Genome-Wide Study of Structural Variants in Bovine Holstein,Montbéliarde and Normande Dairy Breeds

High-throughput sequencing technologies have offered in recent years new opportunities to study genome variations. These studies have mostly focused on single nucleotide polymorphisms, small insertions or deletions and on copy number variants. Other structural variants, such as large insertions or deletions, tandem duplications, translocations, and inversions are less well-studied, despite that some have an important impact on phenotypes. In the present study, we performed a large-scale survey of structural variants in cattle. We report the identification of 6,426 putative structural variants in cattle extracted from whole-genome sequence data of 62 bulls representing the three major French dairy breeds. These genomic variants affect DNA segments greater than 50 base pairs and correspond to deletions, inversions and tandem duplications. Out of these, we identified a total of 547 deletions and 410 tandem duplications which could potentially code for CNVs. Experimental validation was carried out on 331 structural variants using a novel high-throughput genotyping method. Out of these, 255 structural variants (77%) generated good quality genotypes and 191 (75%) of them were validated. Gene content analyses in structural variant regions revealed 941 large deletions removing completely one or several genes, including 10 single-copy genes. In addition, some of the structural variants are located within quantitative trait loci for dairy traits. This study is a pan-genome assessment of genomic variations in cattle and may provide a new glimpse into the bovine genome architecture. Our results may also help to study the effects of structural variants on gene expression and consequently their effect on certain phenotypes of interest.     

Changes in microbial and soil properties following amendment with treated and untreated olive mill wastewater

We investigated the effect of untreated and biologically treated olive mill wastewater (OMW) spreading on the soil characteristics and the microbial communities. The water holding capacity, the salinity and the content of total organic carbon, humus, total nitrogen, phosphate and potassium increased when the spread amounts of the treated or untreated OMW increased. The OMW treated soil exhibited significantly higher respiration compared to the control soil. However, the C-CO2/C(tot) ratio decreased from 1.7 in the control soil to 0.5 in the soil amended with 100 m3 ha(-1) of untreated OMW. However, it slightly decreased to 1.15 in the soil amended with 400 m3 ha(-1) of treated OMW. The treated OMW increased the total mesophylic number while the number of fungi and nitrifiers decreased. Actinomycetes and spore-forming bacteria were neither sensitive to treated nor to untreated OMW. The total coliforms increased with higher doses of treated and untreated OMW. A toxic effect of the untreated OMW appeared from 100 m3 ha(-1). This toxicity was more significant with 200 m3 ha(-1), where microflora of total mesophilic, yeasts and moulds, actinomycetes, and nitrifiers were seriously inhibited except for total coliforms and spore-forming bacteria.     

High Risk HPV Contamination of Endocavity Vaginal Ultrasound Probes: An Underestimated Route of Nosocomial Infection?

Background

Endocavity ultrasound is seen as a harmless procedure and has become a common gynaecological procedure. However without correct disinfection, it may result in nosocomial transmission of genito-urinary pathogens, such as high-risk Human Papillomavirus (HR-HPV). We aimed to evaluate the currently recommended disinfection procedure for covered endocavity ultrasound probes, which consists of “Low Level Disinfection” (LLD) with “quaternary ammonium compounds” containing wipes.

Methods

From May to October 2011 swabs were taken from endovaginal ultrasound probes at the Gynecology Department of the Lyon University Hospital. During the first phase (May–June 2011) samples were taken after the ultrasound examination and after the LLD procedure. In a second phase (July–October 2011) swab samples were collected just before the probe was used. All samples were tested for the presence of human DNA (as a marker for a possible transmission of infectious pathogens from the genital tract) and HPV DNA with the Genomica DNA microarray (35 different HPV genotypes).

Results

We collected 217 samples before and 200 samples after the ultrasound examination. The PCR was inhibited in two cases. Human DNA was detected in 36 (18%) post-examination samples and 61 (28%) pre-examination samples. After the ultrasound LLD procedure, 6 (3.0%) samples contained HR-HPV types (16, 31, 2×53 and 58). Similarly, HPV was detected in 6 pre-examination samples (2.7%). Amongst these 4 (1.9%) contained HR-HPV (types 53 and 70).

Conclusion

Our study reveals that a considerable number of ultrasound probes are contaminated with human and HR-HPV DNA, despite LLD disinfection and probe cover. In all hospitals, where LLD is performed, the endovaginal ultrasound procedure must therefore be considered a source for nosocomial HR-HPV infections. We recommend the stringent use of high-level disinfectants, such as glutaraldehyde or hydrogen peroxide solutions.     

Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal-recessive spastic paraplegia, including Kjellin syndrome

Hereditary spastic paraplegias (HSPs) are genetically and phenotypically heterogeneous disorders. Both "uncomplicated" and "complicated" forms have been described with various modes of inheritance. Sixteen loci for autosomal-recessive "complicated" HSP have been mapped. The SPG15 locus was first reported to account for a rare form of spastic paraplegia variably associated with mental impairment, pigmented maculopathy, dysarthria, cerebellar signs, and distal amyotrophy, sometimes designated as Kjellin syndrome. Here, we report the refinement of SPG15 to a 2.64 Mb genetic interval on chromosome 14q23.3-q24.2 and the identification of ZFYVE26, which encodes a zinc-finger protein with a FYVE domain that we named spastizin, as the cause of SPG15. Six different truncating mutations were found to segregate with the disease in eight families with a phenotype that included variable clinical features of Kjellin syndrome. ZFYVE26 mRNA was widely distributed in human tissues, as well as in rat embryos, suggesting a possible role of this gene during embryonic development. In the adult rodent brain, its expression profile closely resembled that of SPG11, another gene responsible for complicated HSP. In cultured cells, spastizin colocalized partially with markers of endoplasmic reticulum and endosomes, suggesting a role in intracellular trafficking.     

The effect of a daytime 60-min nap opportunity on postural control in highly active individuals

Although napping is commonly used as a strategy to improve numerous physical and cognitive performances, the efficacy of this strategy for improving postural balance has not yet been elucidated. Thus, the aim of this study was to conduct a comprehensive examination of the effect of a 60 min nap opportunity (N60) on different components of postural control. Ten highly active individuals (age = 27 ± 3.5 y, height = 1.75 ± 0.52 m, weight = 66.02 ± 8.63 kg) performed, in a randomized order, two afternoon test sessions following no nap (NN) and N60. Postural balance was assessed using the sensory organisation test (SOT), the unilateral stance test (UST), and the limits of Stability Test performed on NeuroCom^® Smart Balance Master. The subjective rating of sleepiness before and after the nap conditions was also assessed. Compared to NN, N60 improved the composite balance score (p < 0.05, ES = 0.75, Δ = 5.3%) and the average and maximum percentage balance in the most challenging postural conditions of the SOT (p < 0.05 for SOT-4 and 5 and p < 0.0005 for SOT-6; ES range between 0.58 and 1.1). This enhanced postural balance in N60 was accompanied with improved visual (p < 0.05; ES = 0.93; Δ = 8.9%) and vestibular (p < 0.05; ES = 0.81; Δ = 10.5%) ratios and a reduced level of sleepiness perception (p < 0.001, ES = 0.87). However, no significant differences were found in any of the UST and LOS components’ scores (p > 0.05). Overall, a 60 min post lunch nap opportunity may be viable for improving static balance, although further work, involving larger samples and more complex motor activities, is warranted.     

Concordance analysis for QTL detection in dairy cattle: a case study of leg morphology

Background

The present availability of sequence data gives new opportunities to narrow down from QTL (quantitative trait locus) regions to causative mutations. Our objective was to decrease the number of candidate causative mutations in a QTL region. For this, a concordance analysis was applied for a leg conformation trait in dairy cattle. Several QTL were detected for which the QTL status (homozygous or heterozygous for the QTL) was inferred for each individual. Subsequently, the inferred QTL status was used in a concordance analysis to reduce the number of candidate mutations.

Methods

Twenty QTL for rear leg set side view were mapped using Bayes C. Marker effects estimated during QTL mapping were used to infer the QTL status for each individual. Subsequently, polymorphisms present in the QTL regions were extracted from the whole-genome sequences of 71 Holstein bulls. Only polymorphisms for which the status was concordant with the QTL status were kept as candidate causative mutations.

Results

QTL status could be inferred for 15 of the 20 QTL. The number of concordant polymorphisms differed between QTL and depended on the number of QTL statuses that could be inferred and the linkage disequilibrium in the QTL region. For some QTL, the concordance analysis was efficient and narrowed down to a limited number of candidate mutations located in one or two genes, while for other QTL a large number of genes contained concordant polymorphisms.

Conclusions

For regions for which the concordance analysis could be performed, we were able to reduce the number of candidate mutations. For part of the QTL, the concordant analyses narrowed QTL regions down to a limited number of genes, of which some are known for their role in limb or skeletal development in humans and mice. Mutations in these genes are good candidates for QTN (quantitative trait nucleotides) influencing rear leg set side view.     

Kinetic investigation on aqueous decomposition of 2-chloroethylnitrososulfamide

The kinetics decomposition of 2-chloroethylnitrososulfamides (CENS) was studied in aqueous buffered solutions with pH ranging from 0 to 14. The study was monitored by RP-LC-MS and conventional UV spectrophotometry. The reaction proceeded via a pseudo-first-order kinetic with significant correlation coefficient. The major decomposition products from CENS after incubation in phosphate buffer were isolated and identified by NMR and mass spectrometry. The results indicate that the mechanism pathway involves a denitrosation of the CENS and competitive hydrolysis with nucleophilic attack on the sulfur atom and formation of sulfamate compounds.